基于金纳米颗粒标记非洲猪瘟病毒p30蛋白免疫层析检测方法的建立  

Development of a gold nanoparticle labeled immunochromatographic test strip for the detection of African swine fever virus antibodies

作　　者：孙燕燕[1,2] 赵亚茹 李昕 杨吉飞[1] 牛庆丽[1] 林密[1] 王兆贵 刘猛 关贵全[1] 殷宏[1,3] 蒋韬 刘志杰[1] SUN Yan-yan;ZHAO Ya-ru;LI Xin;YANG Ji-fei;NIU Qing-li;LIN Mi;WANG Zhao-gui;LIU Meng;GUAN Gui-quan;YIN Hong;JIANG Tao;LIU Zhi-jie(State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2020年第8期939-945,共7页Chinese Veterinary Science

基　　金：甘肃省重点研发计划项目(18YF1WA131);中国农业科学院基本科研业务费专项(Y2019YJ07-02);农业科技创新工程项目(CAAS-ASTIP-2016-LVRI);国家重点研发计划项目(2017YFD0502300)。

摘　　要：为建立一种用于非洲猪瘟病毒(ASFV)抗体的快速检测方法,本研究将特异性高、抗原性强的重组ASFV p30蛋白用金纳米颗粒标记,制成捕获金标抗原;将p30蛋白及其多克隆抗体(ASFV-p30-Ab)包被在硝酸纤维素膜(NC)上,分别作为检测线(T线)和质控线(C线),制备ASFV p30金纳米颗粒免疫层析抗体检测试纸条,并对该方法开展了初步评价。结果表明,该试纸条可在5-10 min完成检测;以健康猪血清、口蹄疫病毒(FMDV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV-2)等阳性血清作为对照,该方法仅特异性的检出ASFV感染的血清阳性样品,与各对照组血清无交叉反应;以进口ASFV抗体ELISA检测试剂盒作为金标准对阴、阳性样品进行对比检验,该试纸条检出的敏感性和特异性分别为92.52%和92.00%,说明本研究制备的ASFV p30抗体金纳米颗粒检测试纸条具有较高的敏感性和特异性,具备操作简便、检测快速和结果判断容易等特点,具有ASF现场检测的实际应用价值。In order to development of a rapid African swine fever virus(ASFV) antibody test,the recombinant ASFV p30 protein with a merit of specificity and antigenicity was labelled with gold nanoparticles,and used as antigen to establish a colloidal gold immunochromatographic test strip for rapid detection of ASFV antibodies.The recombinant p30 protein and its antibodies were blotted on nitrocellulose membrane as a test line and a control line,respectively.The specificity of the test was assessed by the testing known positive serum against ASFV,and the positive sera against FMDV,CSFV,PRRSV and PCV-2 as well as the serum from a healthy pig as negative control.The results showed that the test strip had detected the ASFV positive serum specifically,while no cross reaction with the controls.The comparison tests of the same group of sera using an INgenasa PPA Compac Blocking ELISA as golden standard showed that the sensitivity and specificity of the test strip were 92.52% and 90.00%,respectively.The strip test was rapid and it could be accomplished with testing serum samples in 5—10 minutes.In conclusion,the colloidal gold immunochromatographic test strip established in the present study showed high specificity and sensitivity,and the test strip with the merits of being rapid,easy-to-operate and easy-to-judge the results with naked eye,so it will be a useful tool for ASFV infection in field test.

关 键 词：非洲猪瘟病毒 p30蛋白 抗体检测 金纳米颗粒 免疫层析法 

分 类 号：S852.659.1[农业科学—基础兽医学]